Electrochemilumiscence method for detecting analytes

ABSTRACT

A method for analyzing a liquid test sample by electrochemiluminescence. The method includes at least one specific biochemical binding reaction that leads to the formation of a complex which contains a chemiluminescence marker and the binding of the complex to a magnetic microparticle. Detection is carried out in a measuring cell having a working electrode in order to determine the concentration of the marked microparticle. The detection cycle includes a purification step, a conditioning step, a recovery step and a measuring step. A specified potential profile is applied to the working electrode during these steps. Between the conditioning step and the recovery step, an additional potential pulse with an oxidizing and/or a reducing potential is inserted into the voltage shape of the detection cycle in order to improve the deposit of the microparticle.

The invention refers to a method for the analysis of a sample withregard to a substance contained therein.

The analysis of a liquid sample is generally concerned with thedetermination of the concentration of a substance (analyte) containedtherein (quantitative analysis). In some cases, it is sufficient simplyto determine whether the analyte is present (in a concentrationexceeding a threshold value) in the sample or not (qualitativeanalysis). In medical applications for which the present invention is ofparticular importance, the analysis of body fluids (primarily blood,blood serum and urine) with regard to the analytes contained therein,such as hormones, antibodies, antigens or drugs, plays an importantrole.

The invention refers to the improvement of a certain type of analyticprocedure which may be designated as an electrochemiluminescence bindingreaction analysis (subsequently referred to as ECL-BBA standing forelectrochemiluminescence biochemical binding analysis). Such a methodhas the following characteristic features.

a) The analytic selectivity is based on a specific biochemical bindingreaction using biochemical substances which selectively can only bind toeach other. Primary examples are immunological chemical bindingreactions between antibodies and antigens or haptens with which theantibodies bind specifically. Other biochemical binding reactions areprotein binding, in particular between avidine and biotin, the lectinecarbohydrate binding, binding between receptors and ligands and thehybridization of nucleic acids.

Such specific biochemical binding reactions have been used for some timefor analytic purposes. There are a plurality of differing one ormulti-step reaction processes (test protocols) which finally lead,through the participation of the analyte and at least one specificallybinding substance contained in the reagent system (binding reagents), tothe formation of a complex characteristic for the analysis. This complexnormally (but not necessarily) contains the analyte.

b) In order to render the complex, whose concentration constitutes ameasure of the analytic result sought, detectable, a marking substances(label) is normally used which is coupled to a binding reagent of thereagent system, e.g. an antibody. The species comprising the markingsubstance and the binding reagent is designated as a conjugate.

The invention refers to cases in which the marking substance is capableof effecting an ECL-reaction. When such a substance is subjected to asuitable electrical potential on a voltametric electrode, it emits lightwhich can be measured photometrically. A second electrochemically activesubstance, designated as a precursor, normally contributes to thisreaction. In practice, primarily a ruthenium complex (ruthenium-tris[bipyridyl]) is used as ECL-label in combination with TPA(tripropylamine) as precursor. The two electrochemically activesubstances react on the electrode each releasing an electron and therebyforming a strongly reducing or oxidizing species. The subsequent redoxreaction brings the ECL-label into an excited state from which itreturns to the ground state with the emission of a photon. The ECL-labelreaction is preferably a circular reaction so that a single labelmolecule emits a plurality of photons after application of a voltage tothe electrode.

c) In the methods to which the invention refers, the ECL-marked complexmolecules characteristic for the analysis are fixed to magneticmicroparticles (beads). In practice, magnetized polystyrol balls havinga diameter of typically 2 to 3 μm are used. Fixing is effected by meansof a pair of specific biochemical binding partners. The pairstreptavidin biotin has turned out to be particularly advantageous. Thebeads are coated with a streptavidin polymer. Biotin is bound to thecomplex molecule.

The beads with the bound marked complex are introduced into themeasuring cell of a measuring apparatus. The cell is equipped theelectrodes (normally a working electrode, a counter electrode and, inparticular for the case of a potentiometric measurement scheme, areference electrode) which are, necessary for generating the electricalfield required for triggering the ECL-reaction. The beads are drawn ontothe surface of the working electrode in the magnetic field of a magnetdisposed below the working electrode. Since this normally occurs inflow-through cells with continuously flowing sample fluids, the magneticdeposition of the beads is designated as “capturing”.

Generally after the capturing step a washing step is carried out duringwhich a washing fluid flows by the working electrode to remove unwantedcomponents. An electric potential required for triggering theECL-reaction is then applied to the working electrode and the resultingluminescence light is measured using a suitable optical detector. Theintensity of the luminescence light is a measure for the concentrationof the marked beads on the surface of the working electrode which, inturn, is a measure of the concentration of the analyte in the sample. Acalibration allows calculation of the sought concentration from themeasured luminescence signal.

A plurality of different variations of this type of ECL-BBA-method havebeen discussed and described in the literature. Such variations mayrefer to each of the individual aspects mentioned.

With regard to aspect a), the tests are distinguished from each other bydifferent test protocols (for example sandwich tests and competitivetests, each with a plurality of different sub-variations). A fundamentaldifference obtains between homogeneous tests which do not requireseparation between the formed complex molecules and the non-complexedconjugate and heterogeneous tests which require such a bound/freeseparation. The present invention can be used for very differing testprotocols as long as they include a reaction sequence which comprises atleast one specific chemical binding reaction and which leads to theformation of a complex which is characteristic of the analysis and whichis marked with an ECL-label.

Also with regard to aspect b) the invention is universally applicable,i.e. it is independent of the ECL-label used and possible additionalcomponents of the ECL-reaction. The invention has turned out to beparticularly usefully for test methods using the mentioned rutheniumcomplex in combination with TPA.

With regard to aspect c), the invention is solely directed to tests inwhich the complex characteristic for the analysis is bound to magneticmicroparticles and in which these microparticles are deposited on thesurface of a working electrode in the magnetic field of the magnet. Theinvention is otherwise independent of variations of aspect c) and cane.g. be used with differing bead materials and sizes as well asdiffering methods for fixing the complex to the beads.

More detailed information concerning the ECL-BBA-method can be takenfrom the extensive literature. Towards this end in particular thefollowing publications are cited, the complete disclosure of which ishereby incorporated by reference:

1) G. F. Blackburn et al. “Electrochemiluminescence Detection forDevelopment of Immunoassays and DNA Probe Assays for ClinicalDiagnostics”, Clin. Chem. 37 (1991), 1534-1539

2) J. K. Leland and M. J. Powell: “Electrogenerated Chemilumenescence:An Oxidative-Reduction Type ECL Reaction Sequence using TriprolylAmine”, J. Electrochem. Soc., 137(1990), 3127-3131

3) J. H. Kenten et al.: “Improved Electrochemiluminescent Label for DNAProbe Assays: Rapid Quantitative Assays of HIV-1 Polymerase ChainReaction Products”, Clin. Chem. 38 (1992), 873-879

4) N. R. Hoyle: “The Application of Electrochemiluminescence toImmunoassay-based Analyte Measurement” in “Bioluminescence andChemilumenescense”; Proceedings of the 8^(th) International Symposium onBioluminescence and Chemilumenescence, Cambridge, September 1994, A. K.Campbell et al. (edit.), John Wiley & Sons

5) WO 89/10551

6) WO 90/11511

As mentioned, the measurement of the ECL-light is normally carried outin a flow-through measurement cell. The cell comprises a narrow flowchannel for the sample fluid, and the working electrode is disposed onone of the walls defining the flow channel. In order to be able tosequentially measure differing samples with the same measuring cell, thecell, in particular the working electrode, must be cleaned betweenmeasurements to remove the beads and other impurities deposited thereon.This cleaning process must be rapid and efficient in order to guaranteea high throughput for the analysis apparatus and good analysisprecision.

Cleaning is therefore not only done physically (passage of air bubbles)and chemically (passage of a cleaning fluid containing, inter alia, adetergent). Rather also electrochemical cleaning takes place byapplication of a strongly oxidizing and/or reducing potential to theworking electrode. The potential is normally sufficiently high that gasbubbles are formed on the surface of the working electrode. Thiseffectively supports the cleaning process. The electrochemicalequilibrium of the electrode surface is, however, so strongly perturbedthat after the cleaning step a conditioning step must be carried out inwhich a sequence of pulses are applied to the working electrode whichcover the entire working potential range of the electrode material used.

Thus, a detection cycle is carried out in the cell which includes asequence comprising a cleaning step, a conditioning step, a capturingstep and a measuring step. During the cleaning step and during theconditioning step a cleaning fluid and a conditioning fluid respectivelyare located in the cell. The sample fluid with the beads is introducedinto the flow-through measuring cell only at the beginning of thecapturing step. Heterogeneous tests comprise an additional washing stepbetween the capturing step and the measuring step. The detection cycleis explained in more detail in references 1 through 6, primarily in WO89/10551.

The ECL-BBA-method is distinguished, in comparison to other analysismethods which are based on the specific binding of biochemical bindingpartners, by simple handling, high sensitivity, a large dynamic range ofmeasurable concentrations, an economical analysis, and good automationpossibilities (by means of corresponding analysis apparatus).

In order to achieve a further increase of the analytical precision ofECL-BBA-methods of the above mentioned type an additional potentialpulse having an oxidizing and/or reducing potential is introduced, inaccordance with the invention, into the voltage curve of the detectioncycle between the conditioning step and the capturing step to improvedeposition of the microparticles, wherein the additional potential pulseis returned to a neutral (neither oxidizing nor reducing) potentialbefore the working electrode is contacted by the sample.

The dependence of the quality of the analysis on the voltage curveapplied to the working electrode during the detection cycle is discussedin WO 89/10511 (reference 5). According to this reference, a constantpotential value, designated as a “preoperative potential”should beapplied at the end of the conditioning step in order to improve thereproducibility of the analysis result. This preoperative potentialshould remain constant until the working electrode is contacted by thesample fluid and the ECL-measurement is carried out. The preoperativepotential should be either an oxidizing potential or a reducingpotential in dependence on the material of the working electrode and onthe electrolyte used.

In the context of the invention it has been discovered that, in contrastto the teaching of reference 5, a substantial improvement concerning theeven deposition of the beads on the surface of the working electrode andthereby an improvement in the reproducibility and precision of theanalysis can be achieved if the additional potential pulse is introducedinto the voltage curve of the detection cycle. It is important that thispulse on the one hand attains an oxidizing or reducing potential valueand on the other hand is returned to a neutral (neither oxidizing norreducing) potential before the sample is introduced into the measuringcell and contacts the working electrode.

While the preoperative potential of WO 89/10551 is intended to influencethe components of the sample pertinent to the generation of theECL-signal, the invention achieves a substantial improvement by means ofan additional electrochemical preprocessing of the electrode. The factthat this leads to an improvement in the deposition of the beads isunexpected, since it was to be assumed that the bead distributiondepends on the properties of the magnetic field, whereas theelectrochemical conditioning and cleaning measures serve for improvementof the signal generation.

A potential pulse as used in the invention is a transient change of thevoltage applied to the working electrode during which an oxidizing orreducing potential value is reached, whereas the electric potential isin the neutral region before and after the potential pulse. The detailedshape of the potential curve may vary. It is in particular not necessarythat the potential curve have a defined geometrical shape (e.g.rectangular, triangular or step function).

An oxidizing potential is an electric potential of the working electrodeby which the surface thereof which is in contact with the conditioningfluid is oxidized. A potential is reducing when it effects anelectrochemical reduction of the metallic surface of the workingelectrode in contact with the conditioning fluid. A potential by whichpractically no oxide or hydride layer is formed on a clean metallicsurface is neutral. This state is also called the double-layer region ofthe corresponding metal.

No generally valid numerical values for the maximum potential of theadditional pulse and for the value to which this potential must bereturned can be given, since these potential values depend on thematerial of the working electrode, on the reference electrode to whichthe potential refers, and (to a lesser extent) on the composition of theconditioning liquid. Those skilled in the art can take more detailedinformation in this regard from published data in particular fromcyclovoltamograms of the electrode material used. In any event, thevalues for an oxidizing, reducing, or neutral potential can bedetermined experimentally.

The invention is explained more closely below with regard to anembodiment schematically represented in the figures.

FIG. 1 shows a schematic representation of the detection unit of anECL-BBA analysis apparatus,

FIG. 2 shows a graphical representation of the time dependence of thepotential applied to the working electrode during a detection cycle,

FIG. 3 shows a cyclovoltamogram of a platinum electrode.

The detection unit 1 shown in FIG. 1 constitutes that part of theanalysis apparatus which automatically carries out the detection cycle.In addition, such an analysis apparatus has units for carrying out thereaction sequence which leads to formation of a complex marked with anECL-label (the concentration of which is characteristic for theanalysis) and to its binding to magnetic microparticles. Thesecomponents are not shown in FIG. 1.

The heart of the detection unit 1 is an electrochemical flow-throughcell 2 in which a working electrode 4 and a counter electrode 5 aredisposed at a narrow flow-through channel 3 in such a manner that theyare contacted by a liquid flowing through the flow-through channel 3.The counter electrode 5 is, as shown, preferably positioned across fromthe working electrode 4 (i.e. at the opposite side of the flow-throughchannel 3). A reference electrode 7 is normally located at the liquidconduit of the detection unit 1, designated in its entirety with 8,outside of the flow-through channel 3. A precision reciprocating pump 9is normally used for suctioning the liquid and is installed in theliquid conduit 8 downstream of the flow-through cell.

A plurality of liquid containers are connected to the conduit 8 upstreamof the flow-through cell 2 from which liquid can be selectivelysuctioned into the flow-through cell 2 as controlled, e.g. by a multipleway valve 10. In the example shown, these are a container 12havingcleaning liquid, a container 13 having conditioning liquid, and acontainer 14 having sample liquid. The sample liquid container 14 isnormally configured as a test tube (reaction tube) which is seatedwithin a processing rotor 15 in which also the steps required forcarrying out the binding reaction are performed. For reasons of clarity,only one of the reaction tubes seated in the processing rotor 15 isshown.

A magnet 17 is disposed in the flow-through measuring cell 2 at the sideof the working electrode 4 facing away from the flow-through channel 3.In the example shown, a permanent magnet is used which can be moved bymeans of a movement mechanism 18 from the capturing position shown (inwhich at least one of its pole pieces is as close as possible to theworking electrode 4) into a neutral position removed from the workingelectrode 4. An electromagnet which can be switched on and off canalternatively be used.

A photo-multiplier is used as a light detector 19 and is positioned onthat side of the flow-through channel 3 lying across from the capturingsurface 4 a in such a manner that its light sensitive surface extendsparallel to the capturing surface 4 a of the working electrode 4 facingthe flow-through channel 3 (and facing away from the magnet 17).

The elements of the detection unit 1 are connected to an electronicsunit 16, provided for controlling the apparatus and processing thesignals from the light detector 19 (the connecting cables are onlypartially shown).

It is important for the function of the detection unit 1 that the timedependence of the potential applied to the working electrode 4(relative, in each case, to the liquid in contact therewith and therebyrelative to the reference electrode) during the individual processingsteps of a detection cycle have particular features which will now bedescribed in more detail on the basis of the graphical representation ofsuch a potential curve shown in FIG. 2. The figure relates to anembodiment having a platinum working electrode. The voltage values Ugiven on the ordinate are measured relative to an Ag/AgCl-referenceelectrode and plotted against the time t in seconds. The detection cycleis repeated in the same manner for each analysis. The followingdescription begins with the cleaning step.

A cleaning step 20 is carried out in each case following the precedingmeasurement in order to free the deposit surface 4 a of the workingelectrode 4 from beads bonding thereto and other impurities or changesin the electrode surface. A strongly oxidizing and/or reducing potentialCl0 or ClR respectively is applied to electrochemically assist thecleaning process. Its potential value is generally higher than that ofall other potentials in the detection cycle (both oxidizing andreducing). An oxidizing potential C10 is preferred —as shown —whosevalue exceeds a likewise oxidizing potential of the precedingmeasurement step 21. In the example shown, the cleaning step includestwo smaller reducing potentials ClR1 and ClR2. This is, however, notabsolutely necessary. The invention is suitable for all detection cyclesin which during the cleaning step an oxidizing or reducing potential isapplied to the working electrode which is so strong that a subsequentconditioning step is required to reestablish electrochemicalequilibrium. A pump 9 feeds during the entire cleaning step cleaningfluid out of the container 12 through the conduit 8 and thereby alsothrough the flow-through channel 3.

A subsequent conditioning step 22 serves to reestablish the requiredelectrochemical equilibrium on the electrode surface following thecleaning step 20. Towards this end, a sequence of alternating reducingand oxidizing potential pulses are applied to the working electrodedesignated in FIG. 2 with C01, CR1, CO2, CR2, CO3 and CR3.

The sequence of conditioning pulses preferably comprises —as shown —analternating sequence of oxidizing and reducing pulses, an even totalnumber of reducing and oxidizing pulses being particularly preferred.The duration of each conditioning pulse is, in practice, less than 1second. Values of less than 0.7 seconds and more than 0.1 seconds arepreferred and the range between approximately 0.3 seconds andapproximately 0.5 seconds has turned out to be particularly useful.

The invention is characterized by the introduction of an additionalpotential pulse into the voltage curve of the detection cycle,designated in FIG. 2 with DIP for “deposition improvement pulse”. It isintroduced after the conditioning step 22 and before the point of timedesignated in FIG. 2 as t_(p) at which the sample is pumped out of thesample container 14 via the valve 10 into the flow-through channel 3 (sothat the beads contained in the sample fluid are brought into contactwith the working electrode 4). This additional pulse is high enough toattain an oxidizing or reducing potential value for a very short timeduration which is nevertheless sufficient to electrochemically influencethe electrode surface (preferably at least approximately 0.05 secondsand particularly preferred at least 0.1 seconds) . It is furthermoreimportant that the potential be returned to a neutral potential value(neither oxidizing nor reducing) before time t_(p). The example of anoxidizing DIP shown, wherein it follows a preceding reducing potentialpulse CR of the conditioning step 22, is particularly preferred.

Within the framework of the invention, it has been discovered that bymeans of the DIP the surface of the electrode is prepared in an optimalmanner for the capturing step. Simultaneously the surface is adapted tothe properties of the beads (e.g. surface properties, zeta potential,stickiness etc.). Experimental optimization of the DIP allows adjustmentof the deposition pattern such that it optimally corresponds to therequirements of a ECL-detection procedure.

At time t_(p) the magnet 17 is located in the capturing position shownin FIG. 1. Attracted by its magnetic field the microparticles flowingwith the sample fluid through the flow-through channel 3 are depositedon the surface of the working electrode. During the capturing step 23and also during the subsequent washing step 24 the working electrodepotential is preferably —as shown —in the neutral range. In conventionalmethods, the working electrode is normally during these steps separatedfrom the potentiostat and therefore does not have a defined potential.In accordance with WO 89/10551, the “preoperative potential”, describedtherein, namely a constant oxidizing or reducing potential, should beapplied to the working electrode 4 during this part of the detectioncycle.

Aside from the potential of the working electrode, the capturing step23, the washing step 24 and the subsequent measuring step 21 are carriedout in conventional manner. The multiple way valve 10 is switched attime t_(w) such that, instead of the sample liquid, the conditioningliquid which simultaneously serves as washing liquid for the bound/freeseparation is suctioned out of the container 13 and into the conduit 8.

Instead of the one additional potential pulse DIP, a plurality ofadditional potential pulses can also be introduced into the voltagecurve of the detection cycle between the conditioning step 22 and thecapturing step 23, in which case all DIPs are preferably eitheroxidizing or reducing. The total duration of time during which the oneDIP or the plurality of DIPs are located in the oxidizing or reducingregion should be at least 0.05 seconds in each detection cycle,preferably at least 0.1 seconds, and at most 1 second and preferably atmost 0.3 seconds.

FIG. 3 shows a cyclic voltamogram of a platinum electrode. These typesof measurements are usually carried out by varying the potential appliedto the electrode, relative to a reference electrode, in a triangularfashion to thereby generate the shown current/voltage curves. The cyclicvoltamogram shows results from the measurement of a platinum electrodein contact with a 1M-sulfuric acid solution. The voltage values on theabscissa are relative to a normal hydrogen electrode. The current flow Iin mA/cm² is given along the ordinate.

If one follows the current voltage dependence departing from point A inthe direction of the arrow, the potential is initially located in aregion in which only an amount of current which can hardly be measuredflows, associated with the charging up of a double layer. This region isreferred to in the English language literature as the “double-layerregion”. The current flow increases strongly in curve region B. Hereoxidizing potential values as used in the present invention are reached,i.e. the platinum surface is electrochemically oxidized. The area underthe curve corresponds to the amount of charge needed for the oxidation.

Preferably, the working electrode is a platinum electrode and thehighest value of the additional potential pulse (DIP) corresponds to avoltage of at least 0.6 V, preferably at least 0.8 V, relative to anAg/AgCl reference electrode. Preferably, the voltage is at most 1.6 V,preferably at most 1.2 V, relative to an Ag/AgCl reference electrode.

When the voltage applied to the electrode decreases after achieving itsmaximum value (here approximately 1.5 V), the current flow is initiallylow, but then increases again in the region in which the oxide layer isremoved (curve region C). After the oxide layer has been removed, thecurrent again falls close to zero in the double-layer region until thevoltage reaches a value which causes a reduction of the platinum(previously substantially pure). This rising in the curve region Dindicates a reducing potential value in the sense of the presentinvention. The voltage region between the oxidizing and the reducingpotential is designated as the neural region N. After the potential isreversed again at approximately 0.1 V, the reduction layer on theplatinum surface is removed in curve region E.

EXAMPLE

Comparison tests were carried out with the Elecsys 2010 apparatus ofBoehringer Mannheim GmbH to compare the results with and without DIP.The detection cycle thereby corresponded to FIG. 2. The experiments withDIP inncluded an additional oxidizing pulse of rectangular shape with apule height of +0.9 V and a pulse duration of 0.2 sec. For a PSA test(PSA=prostrate specific antigen) the following values were obtainedwithout DIP.

TABLE 1 Conc. VK No. ng/ml N MW % 1 0.00 11 1020.94 2.66 2 0.75 44957.80 2.00 3 2.97 2 16698.81 1.75 4 16.20 2 84445.07 2.10 5 75.10 2398381.77 0.99 6 142.00 2 741512.39 1.62

With DIP the following values were obtained:

TABLE 2 Conc. VK No. ng/ml N MW % 1 0.00 11 1045.33 1.12 2 0.75 45874.34 0.63 3 2.97 2 2093.29 1.06 4 16.20 2 107626.88 1.26 5 75.10 2497223.62 0.99 6 142.00 2 950228.61 0.26 The column titles are definedas follows: Conc.: nominal concentration of the calibration sample usedN: number of measurements carried out MW: average value of themeasurement signal in arbitrary units VK: variation coefficient of thesignal in percent

One notices that the DIP leads to a substantial improvement in thesignal dynamics (the quotient between the highest and the lowest MW).With DIP, this value is 909 and thereby approximately 25% higher thanwithout DIP (726). In addition, the precision, reflected by themagnitude of the variation coefficient VK, is substantially improved.

By means of visual observation and using video recordings during theanalysis cycle, it has been determined that the beads are also depositedin a substantially more stable manner. Without DIP, the beads moveduring the washing step. This is disadvantageous for the measurementprecision, particularly since it can cause loss of previously depositedbeads from the working electrode.

What is claimed is:
 1. An electrochemiluminescence method of detectingan analyte in a liquid sample, said method comprising: (a) carrying outa reaction sequence comprising at least one specific biochemical bindingreaction to form a complex as a result of the concentration of theanalyte in the liquid sample, said complex comprising a markingsubstance capable of effecting an electrochemiluminescence reaction,said complex further being bound to magnetic microparticles, and (b)carrying out a detection cycle in a measuring cell having a workingelectrode for determination of the concentration of said boundmicroparticles, said detection cycle including a sequence comprising;(i) a cleaning step during which a strongly oxidizing and/or reducingpotential is applied to said working electrode for electrochemicalcleaning of said electrode, (ii) a conditioning step comprising applyinga sequence of a number of alternating reducing and oxidizing potentialpulses to said working electrode, wherein said sequence comprises a lastpulse; (iii) a capturing step during which said sample containing saidmicroparticles is contacted with said working electrode in such a mannerthat said microparticles are attracted by the magnetic field of a magnetpositioned on the side of said working electrode facing away from saidsample, thereby being deposited on a surface of said working electrodefacing said sample, and (iv) a measuring step during which a potentialis applied to said working electrode to trigger saidelectrochemiluminescence reaction and the light emitted by said markingsubstance is measured to determine the concentration of said substanceon said deposit surface of said electrode, thereby determining theanalyte in the liquid sample, said method further comprising applying atleast one additional potential pulse having an oxidizing or reducingpotential into the voltage curve of the detection cycle between saidconditioning step and said capturing step, wherein said additionalpotential pulse is returned to a neutral potential prior to contactingof said working electrode by said sample.
 2. The method of claim 1,wherein the last potential pulse of said conditioning step precedingsaid additional potential pulse is a reducing potential pulse.
 3. Themethod of claim 1, wherein said additional potential pulse is anoxidizing potential pulse.
 4. The method of claim 1, wherein during saidconditioning step, the number of said reducing potential pulses is equalto the number of said oxidizing potential pulses.
 5. The method of claim1, wherein said additional potential pulse has the same polarity as thepotential triggering said electrochemiluminescent reaction during saidmeasuring step and the highest value of said additional potential pulseis less than the highest value of the potential triggering saidelectrochemiluminescent reaction.
 6. The method of claim 1, wherein saidworking electrode is a platinum electrode and the highest value of saidadditional potential pulse corresponds to a voltage of at least 0.6 Vrelative to a Ag/AgCl reference electrode.
 7. The method of claim 1,wherein said working electrode is a platinum electrode and the highestvalue of said additional potential pulse corresponds to a voltage of atleast 0.8 V relative to a Ag/AgCl reference electrode.
 8. The method ofclaim 1, wherein the highest value of said additional potential pulsecorresponds to a voltage of at most 1.6 V relative to an Ag/AgClreference electrode.
 9. The method of claim 1, wherein the highest valueof said additional potential pulse corresponds to a voltage of at most1.2 V relative to an Ag/AgCl reference electrode.
 10. The method ofclaim 1, wherein a plurality of additional potential pulses areintroduced into the voltage curve of the detection cycle between saidconditioning step and said capturing step, wherein the last additionalpotential pulse is returned to a neutral potential prior to contactingof said working electrode by said sample.
 11. The method of claim 1,wherein the total amount of time during which the at least oneadditional potential pulse is in the oxidizing or reducing potentialregion respectively is, in each detection cycle, at least 0.05 seconds.12. The method of claim 1, wherein the total amount of time during whichthe at least one additional potential pulse is in the oxidizing orreducing potential region respectively is, in each detection cycle, atleast 0.1 seconds.
 13. The method of claim 1, wherein the total amountof time during which the at least one additional potential pulse is inthe oxidizing or reducing potential region respectively is, in eachdetection cycle, at most 1 second.
 14. The method of claim 1, whereinthe total amount of time during which the at least one additionalpotential pulse is in the oxidizing or reducing potential regionrespectively is, in each detection cycle, at most 0.3 second.